Guthrie H David, Welch Glenn R
US Department of Agriculture, Animal Bioscience and Biotechnology Laboratory, Agricultural Research Service, Beltsville, Maryland, USA.
Methods Mol Biol. 2008;477:89-97. doi: 10.1007/978-1-60327-517-0_8.
A flow cytometric method was developed to identify viable, energized sperm cells with high mitochondrial inner transmembrane potential (Deltapsi(m)), >80-100 mV using the mitochondrial probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and the impermeant nuclear stain propidium iodine (PI). This flow cytometric method is described in detail here. When in contact with membranes possessing a high Deltapsi(m), JC-1 forms aggregates (J(agg)) that are fluorescent at 590 nm in response to 488 nm excitation. We found that the reactive oxygen species generator, menadione reduced sperm motility and reduced Deltapsi(m) in a dose responsive fashion that was closely correlated with the loss of motility.
我们开发了一种流式细胞术方法，使用线粒体探针5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑基羰花青碘化物（JC-1）和非渗透性核染料碘化丙啶（PI）来鉴定具有高线粒体内膜跨膜电位（Δψm）（>80-100 mV）的活的、有活力的精子细胞。本文详细描述了这种流式细胞术方法。当与具有高Δψm的膜接触时，JC-1会形成聚集体（J(agg)），在488 nm激发下，该聚集体在590 nm处发出荧光。我们发现，活性氧产生剂甲萘醌以剂量反应方式降低精子活力并降低Δψm，这与活力丧失密切相关。
